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Vol. 260,No. 21, Issue of September 25,pp. 11461-11467, 1985

(01985 by The American Society of Biological Chemists, Inc Printed in U.S. A.

Signal Transduction and Ligand-Receptor

Dynamics in the
Human Neutrophil

(Received for publication, July 27, 1984)

Larry A. Sklar, PaulA. Hyslop, Zenaida G. Oades, Geneva M. Omann, Algirdas J. Jesaitis, Richard
G. Painter$, and CharlesG. Cochrane
From the Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037

The responses of neutrophils to formyl peptides are Neutrophils or polymorphonuclearleukocytes are stimu-
initiated and in many eases achieve a maximal level lated by specific ligands to participate inprocesses which
prior to equilibrium receptor occupancy. In order to contribute to inflammation and host defense. The N-formyl
begin to understand the linkagebetween receptor oc- peptidereceptorhasproven t o be a particularly valuable
cupancy and cell response we have used a pulsed bind- startingpoint in elucidatingthe molecularmechanism of
ing procedure to analyze: 1) the number of receptors neutrophil activation for several reasons. First, stimulatory
contributing to three potential signalling events and and inhibitory ligands with characterized structure-function
six functional responses and 2) the evolution of these relationships are available (1, 2). Second, radiolabel, fluores-
responses once ligand binding is interrupted. We find cent label, and photoaffinity label derivatives of several of
that thehalf-optimal elevationsof the potential signals these ligands have been characterized(3-8). Third, the occu-
are produced by 4 % occupancy (Ca”’) or 1-3% occu-
pancy of receptors by these ligands leadsto several cell
pancy (CAMP, membrane depolarization). In contrast,
actin polymerization and a rapid light scatter response functions which includefreeradical generation, secretion,
are elicited by <O. 1% occupancy. Half-optimal elastase production of autocoids such as leukotrienes and other prod-
release and degranulation require -3% occupancy. ucts of arachidonic acidmetabolism, andchemotaxis (see
While half-optimal 0;production and aggregation re- reviews, Refs. 9-11). Finally, efforts by many investigators
quire -30% occupancy, the half-optimal rate of 0; have already succeeded in identifying a number of potential
production requires less than 10%occupancy. second messengers such as Ca2+, cyclic nucleotides, inositol
To resolve the apparentlack of correlation between trisphosphate, diacylglycerol, etc.
the responses and the signalswe examined their time In order toprobe the relationships between ligand binding
courses following the pulse of stimulation. At least four and cell response, we described a pulsed occupancy protocol
responses and one signal are transient anddecay while (6, 12) which used a fluoresceinated N-formyl hexapeptide
occupied receptors remain on the membrane surface. (FLPEP’) followed by a high affinity antibody tofluorescein
These include the Quin 2-Ca”’ signal, actin polymeri- to limit furtherligand binding to thereceptor. We suggested
zation, the light scatter response, 0;generation, and thatcompetitiveantagonists (e.g. benzyloxycarbonyl-Phe-
aggregation. Ca2+ elevation is correlated with the re- Met) could be used in the same capacity (6) and Korchak et
sponses in that: 1) each of these responses is transient al. (13) recently used t-BOC-Phe-Leu-Phe-Leu-Phe in this
unless new receptors are occupied; 2) occupancy of capacity.
nearlyall of thereceptorscontributestothe time Several responses have now been qualitatively character-
course of these responses; 3) when binding is inter- ized (6, 12, 13). We found thatthemembranepotential
rupted, the responses decay with a half-time of 15 s,
following a latency of -10 s or Iess (except for disag- sensitive dye response of 3,3‘-dipropylthiocarbocyanine( 5 )
gregation where latency is 30-40 6). We discuss evi- was elicited by a few seconds of ligand binding (near &). If
dence in support of the hypothesis that transient cell the responsewas allowed to achieve its maximal level, its rate
responses arise from transient receptor activation. of recovery was not influenced by the inhibition of ligand
binding. In contrast, the generation of 0; was observed to
depend upon the entire time course of ligand binding. From
fragmentary binding data we estimated that less than 10%
* This work was supported by United States Public Health Service occupancy contributed to the dye response while more than
Grants AI19032, AI17354, and RR0083, by a grant-in-aid from the 30% contribute to 0; production. Subsequently, we showed
American Heart Association with funds contributed in part by the that the extent of degranulation in cytochalasin B-treated
Riverside County, California chapter, by an Established Investigator cells depended upon theperiod of binding aswell as thedose
Award to L. A. S. from the American Heart Association with funds
contributed in part by the California Affiliate, and by a grant from of ligand (12). We estimated that less than 10% of the recep-
Eli Lilly Laboratories. This is publication 34581” from the Re- tors contributed to degranulation.
search Institute of Scripps Clinic. Portions of this work were first
presented at theBiophysical Society Meeting, February 19,1983. The ’The abbreviations used are: FLPEP (fluoresceinated hexapep-
costs of publication of this article were defrayed in part by the tide), N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-ly-
payment of page charges. This article must therefore be hereby sine-fluorescein; antiFL, high affinity polyclonal rabbit antibody to
marked “aduertisement” in accordance with 18 U.S.C. Section 1734 fluorescein; DiS-C3(5), 3,3’-dipropylthiocarbocyanine;PHPA,par-
solely to indicate this fact. ahydroxyphenylacetic acid Quin 2, (2-[(2-bis[carboxylmethyl]amino-
$ Current address: Department of Biochemistry, University of. 5-metbylphenoxy)methyl]-6-aminoquinoline); t-BOC, t-butoxycar-
Texas Health Center, P.O. Box 2003, Tyler, TX 75710. bonyl.

11462 Receptor
Occupancy and Transient PMN Responses
Korchak et at. (13) have extended these observations to TABLEI
include measurements ofCa’+ (using Quin 2, chlortetracy- Fractional receptor occupancyfor several neutrophil responses
cline, 45Ca),degranulation, shape/volume ratio, 0 2 produc- Time of Per cent occupancy
tion, andaggregation, all incytochalasin B-treated cells. They
concluded that cells, are fully committed to the former five
(No’ donors)” zfjz: required to give
half-optimal responseb

responses within a few seconds of binding while the last two nM 8

require continuous occupancy, and based on measurement at Right angle 0.01 (4+) 10 <0.1 (10-20 receptors)
a single concentration of ligand, they indicated that “commit- Ca2+(Quin 2) 50.05 (5+) 5 4 (200 receptors)
ment” of cells to a particular response depended upon the CAMP 1-2 0.1 (5+) 15
length of the binding period (as opposed to the number of “Depolarization” 0.2 (5+) 30 1-4
receptors occupied). They also suggested that continuous oc- Elastase 0.1 (7+) 30 3k2
0;Rate 0.3 30 7-10
cupancy rather than new occupancy is required to support Magnitude 0.3 (>lo) >3 min 25-30
commitment. In their binding studies, with cytochalasin B-
a Values in parentheses indicate the number of occasions on which
treated cells, they observe rapidly and totally reversible bind- full dose-response analysis were undertaken. + indicates additional
ing once the antagonist is added. This binding behavior is occasions when only a limited number of doses were studied.
different from the binding observed at 37 “C in cells which * From pulse experiments.
have not been treated with cytochalasin B (3, 4,14). e From Ref. 25.

There are three issues which need clarification. First, how From Refs. 16 and 18.
many receptors are required for different cell responses?
Table I.4The major findings are thatseveral responses achieve
Second, is continuous or new receptor occupancy required in maximal levels with less than 1%occupancy (light scatter and
commitment of cellular responses? Third, what is thebehav- actin polymerization), several responses achieve maximal lev-
ior of the cellular responses once the ligand binding is inhib-els with occupancy in the range of 1 to 10% of the receptors
ited? (Ca’+, CAMP, membrane potential sensitive dye responses),
In this reportwe describe the dependence of nine elements while 0; and aggregation are maximal following the occu-
of neutrophil response on receptor occupancy under condi- pancy of nearly all of the receptors. Degranulation (the only
tions where cells bind and rapidly internalize ligand. We have response studied in the presence of cytochalasin B) requires
defined occupancy-response relationships for three potential less than 10%occupancy.
biochemical intermediates (Ca”, CAMP,and ion fluxes) and Both the rapid right angle response and F-actin polymeri-
six cellular responses (free radical production, elastase secre-
zation are remarkable in that anear optimal response is
tion and degranulation, cell aggregation, a transient right elicited by 0.01 nM FLPEP. Following exposure to 0.01 nM
angle light scatter response which may reflect membrane FLPEP, roughly 0.2% of the receptors are occupied at 10 s,
ruffling, and F-actin polymerization). We have studied the the time when the maximal response is detected. This means
evolution of cell responses once the binding of peptide to the that no more than about 120 of 60,000 of the receptors are
occupied. From pulse experiments at these low FLPEP con-
cell surface receptors is interrupted. We find that atleast five
cell responses turn off (Quin 2-Ca2+elevation, actin polym- centrations we conclude that even a smaller number of recep-
erization, the scatter response, 0; generation, and aggrega- tors, probably no more than 10 or 20, are involved in initially
tion) when binding is inhibited, but at a time during which eliciting (but not sustaining) this response, indicating that
the receptors are still occupied and predominantly on the the responses have been dramatically amplified.
external surface of the cells. While these responses may Transient Responses-The concept of “optimal response”
achieve optimal level (or rate in the has subtly different implications in the different cell re-
case of OF) due to a small
number of receptors, they require the participation of nearly sponses. In the case of 0; production, total production in-
all of the receptors for the responses to be sustained over thevolves occupancy of all of the receptors; however, a maximal
rate of production requires rapid occupancy of a much smaller
entire time course of cell response. The transient responses
fraction of receptors. Ifnew occupancy is interrupted (see
are interpreted in terms of transient activation of the recep-
below) during the time course of binding, the production rate
tors. is not sustained and the total production is reduced. Aggre-
MATERIALS AND METHODS AND RESULTS’ gation follows a similar pattern. Even though Ca2+elevation,
right angle scatter,andactin polymerization require only
DISCUSSION small levels of occupancy to elicit “optimal levels,” these levels
are not sustained unless binding continues. These transient
Occupancy-Response Relationships in Neutrophils-We responses have qualitatively parallel dependences on occu-
have used a pulse stimulation approach to evaluate the rela- pancy.
tionship between receptor occupancy and neutrophil activa- Degranulation either measured by the elastase or light
tion. The various responses can be classified according to the scatter assays is not transient in the sense that once enzymes
number of receptors required to elicit the optimal response
and thebehavior of the response once binding is inhibited. ‘Two factors influence the accuracyof these estimates: 1) the
The receptor occupancy requirements are summarized in accuracyof the rate constants; and 2) the variability in receptor
numbers among donors. Based on multiple determinations of rate
* Portions of this paper (including “Materials and Methods,” “Re- constants (Ref. 14), the calculated fractional receptor occupancy is
sults,” Figs. 1-9, Table S-I, and Footnote 3) are presented in miniprint accurate to within 30% inthe occupancy response analyses. The
at the end of this paper. Miniprint is easily read with the aid of a variability in receptor numbers (-30,000-120,000 receptors/cell) will
standard magnifying glass. Full size photocopies are available from not affect the fractional occupancy calculated for short times of
the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, binding (as long as ligand is in excess) but will affect the absolute
MD 20814. Request Document No. 84M-2331, cite the authors, and number of receptors occupied compared to a typical donor (50,000-
include a check or money order for $8.40 per set of photocopies. Full 60,000 receptors/resting cell). Wedo not know whether there are
size photocopies are also included in the microfilm edition of the systematic differences in the responsiveness of cells from donors with
Journal that is available from Waverly Press. different receptor numbers.
Receptor Occupancyand Transient PMN Responses 11463
are released, the receptor occupancy is unrelated to the en- responses found here, three have been studied in common.
zyme activity. The membrane potential response is unusual We both agree on the transient behavior of 0; (6,13). They
in that once an optimal response is achieved, the recovery or found that aggregation (in cytochalasin B-treated cells where
turn off of the response is not influenced by the additional there is extensive degranulation) is not reversible. We find
ligand binding (6). There was insufficient resolution in our that aggregation in the absence of extensive degranulation is
cAMP data to determine the behavior of this response once at least partially reversible (Fig. 7). It is likely that released
binding was interrupted. granule proteins under degranulating conditions contribute
Occupancy-Response Analysis and Receptor Heterogeneity- to sustained aggregation. They suggest that cells are commit-
Occupancy-response curves should be based on pulse or ki- ted to a full Ca2+ (Quin 2) response (an optimal response
netic data rather than on steady-state binding data since most sustained over its entire time course) by the first seconds of
responses are elicited prior to equilibrium binding. Based on binding. We find that an optimal response is elicited by a
total 0; production and degranulation (Figs. 5 and 6),we small number of receptors, but that this elevation is not
observe that such curves are initially linear and extrapolate sustained when binding ends. We have examined ligand doses
through the origin. This means that each participating recep- between 0.1 and 1 nM at times up to 60 s (e.g. Fig. 2) and find
tor contributes in similar
a way to theresponses. The behavior that whether antiFL and FLPEP or t-BOC-Phe-Leu-Phe-
of 0; is particularly revealing in that theoccupancy-response Leu-Phe and unlabeled ligand are used the Quin 2 response
curve is approximately linear up to 70% or more of the always decays with similar kinetics after the inhibition of
receptors (within the limitations of these present measure- binding (data not shown).
ments). Thus, from an occupancy standpoint the receptors A Transient Receptor Hypothesis-In principle, the decay
are relatively homogeneous. of the responses following the inhibition of ligand binding
In systems where monovalent ligand interacts with mon- could depend upon either receptor or postreceptor events or
ovalent receptor such as in cyclase activation in hormone- both. There are several mechanisms by which receptors that
sensitive adrenergic receptor systems (31), we expect linear are long-lived in an activated state could, nonetheless, give
occupancy-response because each receptor contributes in a rise to responses that decay oncenew ligand binding is halted.
similar way to the response. When aggregation or dimeriza- 1. Receptors are activated as long as they are occupied, and
tion of occupied receptors plays a role in the activation of the responses decay when binding is inhibited because ligand
other systems, there is a possibility that such cooperativity dissociation is rapid. This explanation seems to be the one
could be manifested in sigmoidal occupancy response (32,33). offered by Korchak et at. (13). However, in our experiments
In the case of cAMP and Ca”, the occupancy-response the decay of the responses comes at a time when receptors
curves up to theoptimal responses can be evaluated (e.g. Fig. remain largely occupied (Fig. l),i.e. within a minute of the
3). It can be estimated that since -PM increases in the cessation of new binding. Since the binding and processing of
concentrations of these species (22, 23, 25, 34) are elicited by ligand is strongly influenced by the presence of cytochalasin
-1% of the receptors (or no more than -1000 receptors/cell) B (asused by those workers) and thegranule enzymes released
that at least 100-1000 of these species are produced by each during degranulation, we believe that total, rapid dissociation
receptor. In the accompanying paper (35) we suggest that a of ligand is uncharacteristic of untreated neutrophils (3, 4,
much larger amount of Ca2+is actually made available, prob- 14).
ably representing in the lower limit an amplification at the 2. Receptors are activated as long as they are occupied, but
receptor of 1000’s of Ca2+per activated receptor. the responses decay when ligand binding is inhibited because
Ligand Binding and Cell Responses during Pulse Experi- the coupling of all receptors (whether occupied or unoccupied)
ments-The extent of ligand binding during pulse experi- to cell response becomes markedly less efficient withtime. The
ments is regulated by the length of time (“pulse width”) and production of superoxide anion (Fig. 6C, or the maintenance
the dose of ligand (“pulse height”). By varying the dose of aggregation, Fig. 7B), is roughly linear with receptor oc-
between 0.01 and 10 nM and thetime from seconds to minutes cupancy up to at least 70%occupancy. This implies that
receptor occupancy can be varied between <0.1 and >90% newly occupied receptors contribute more or less equally to
(Table S - I ) . the cell response. Thus, newly occupied receptors are indeed
Ligand binding during pulse conditions has been examined activated, during the time course of binding, and continue to
byflow cytometry and by spectrofluorescence using either be coupled to cell responses.
antibody to fluorescein or competitor to inhibit ligand bind- 3. Receptors are active as long as they are occupied, but
ing. These different procedures yield comparable results (14). responses decay when new binding is inhibited because the
In this paperwe present a representative binding experiment decay process itself is a stimulated process. I f decay were a
performed under conditions analogous to those used to study stimulated event, wewould expect the rate of decay to be
cell responses to pulses (Fig. 1).The spectrofluorometric assay sensitive to the number of receptors occupied. However, the
was chosen because at present it allows better resolution of decay kinetics of 0; production (Fig. 6B)or Ca2+elevation
dissociability at early time. Once binding is inhibited by (Fig. 2B) are independent of the extentof stimulation, varying
antibody to fluorescein, ligand dissociation with a half-time the number of receptors occupied by altering the period of
of several minutes is observed. binding (as shown) ortheconcentration of stimulus(not
Of the transientresponses, four of five examined here decay shown). Comparable results areobtained for aggregation (Fig.
with a half-time of -15 s (Ca2+, actin,light scatter, and 0;) 7), actin polymerization (Fig. 9), and right angle scatter (30).
and the fifth, disaggregation, requires 30-45 s before it is We believethat decay reflects a constant homeostatic process
detected (Figs. 2,6-9). The decay phases of the responses are independent of receptor occupancy.
complete while receptors are stilloccupied and predominantly These arguments imply that the decay of cell responses
on the membrane surface, prior to internalization. For ex- does not arise from a loss of activated receptor due to ligand
ample, after 1 min of ligand binding at 37 “C, -10% of the dissociation, a loss of coupling ability of newlyoccupied
occupied receptors have been internalized. receptors to the response pathway as a function of time, nor
These decay data have some distinguishing features from to a stimulation of the decay process by the ligand binding
those reported by Korchak et al. (13). Of the five transient event. In order to reconcile all of these observations we
11464 Receptor
and Transient PMN Responses
hypothesize thatresponses decaywhenligand bindingis Showell, H. J., and Becker, E.L. (1982) Biochemistry 21,257-263
3. Sullivan, S. J., and Zigmond, S. H. (1980) J. Cell Biol. 85, 703-711
inhibited because the receptor itself resides in a transiently 4. Niedel, J., Wilkinson, S., and Cuatrecasas, P. (1979) J. Biol. Chem. 254,
activated state. When binding is inhibited, activated receptors 10700-10706
5. Niedel, J. E., Kahane, I., and Cuatrecasas, P. (1979) Science (Wash. D.C.)
disappear rapidly and fail to generate new signals. The cell 205, 1412-1414
responses decay thereafter because no new signals are avail- 6. Sklar, L. A,, Oades, Z. G., Jesaitis, A. J., Painter, R. G., and Cochrane, C.
G. (1981) Proc. Natl. Acad. Sci U. S. A. 78, 7540-7544
able to sustain the activation sequence. The photoreceptor, 7. Painter, R. G., Schmitt, M., Jesaitis, A. J., Sklar, L. A,, Preissner, K., and
rhodopsin, for example, is only transiently active following Cochrane, C. G. (1982) J. Cell. Biochem. 20, 169-180
8. Schmitt. M.. Painter. R. G.. Jesaitis. A. J.. Preissner. K.. Sklar. L. A,. and
theinteractionwithits ligand, thephoton.Itsactivityis
I .

Cochrane,' C. G. (1983) J.' Biol. Chkm. 258,649-654. '

inversely related to itsdegree of phosphorylation (36). 9. Becker, E. L., Naccache, P. H., Showell, H. J., and Walenga, R. W. (1981)
in Lymphokines, Vol. 4, pp. 297-334, Academic Press, New York
Certain limitations can be placed on the putative lifetime 10. Snyderman, R., and Goetzl, E. J. (1981) Science (Wash. D.C.) 213, 830-
Q W.
of the active receptor. Ligand-receptor complexes remain on 11. Weissmann, G., Smolen,J.,Korchak, H., andHoffstein, S. (1981) in
the membrane fora half-time -3 min prior to internalization. Research Monograph in Cell and Tissue Physiology (Dingle, J. T., and
However, many cell functions decay within 30-40 s of the Gordon, J. L., eds) pp. 15-31, Elsevier North-Holland Biomedical Press,
New York
inhibition of ligand binding. The decay of the function may 12. Sklar. L. A,. McNeil. V. M.. Jesaitis. A. J.. Painter. R. G.. and Cochrane. ~~ ~

C. G. (1982) J. Bid. Cheh. 257,5471-5475

imply: 1) the generating system for the function has itself 13. Korchak, H. M., Wilkenfeld, C., Rich, A. M., Radin, A. R., Vienne, K., and
decayed; 2) the signalswhich activate the function-generating Rutherford. L. E. (1984) J. Biol. Chem. 259.7439-7445
14. Sklar, L. A., Finney, b . A , Oades, Z. G., Jesaitjs, A. J., Painter, R. G., and
systems have decayed; and 3) the ligand-receptor complexes Cochrane. C. G. (1984) J. Biol. Chem. 259.5661-5669
which lead to signals are therefore no longer generating sig- 15. Henson, P. M., and Oades, Z. G . (1975) J. Clin. Inuest. 56, 1053-1061
nals. The decay period (30-40 s) could therefore represent an 16. Sklar, L. A., Jesaitis, A. J., Painter, R. G., and Cochrane, C. G. (1981) J.
B i d . Chem. 256,9909-9914
upper limit bothfor the lifetime of active receptor and for the 17. Hyslop, P. A., and Sklar, L.A. (1984) Anal. Biochem. 141,280-286
entire activation sequence. Aggregation may be a special case 18. Sklar, L. A., Oades, Z. G., and Finney,D. A. (19M) J. Immunol. 133,1483-
because part of the sequence of activation of aggregation may 19. Hiward, T. H., and Meyer, W. H. (1984) J. Cell Biol. 98, 1265-1271
Tsien, R. Y., Pozzan, T., and Rink, T. J. (1982) J. Cell B i d . 94, 325-334
involve actin polymerization and that cells begin to disaggre- 20.
21. Atkinson, J. P., Kelly, T. P., Weiss, H. J., Wedner, J. F., and Parker, C.
gate only after 30-40 s, a time when actin hasdepolymerized W. (1978) J. Immunol. 121,2282-2291
22. Simchowitz, L., Fishbein, L. C., Spilherg, I., and Atkinson, J. P. (1980) J.
(Fig. 9 and Ref. 30). Immunol. 124, 1482-1491
If one examines the recovery phase in more detail it is 23. Seligmann, B. E., and Gallin, J. I. (1983) J. Cell. Physiol. 115, 105-115
apparent that only a few seconds elapse (<lo s) between the 24. Smolen, J. E., and Weissmann, G. (1981) Biochim. Biophys. Acta 672,
time that binding has stopped and cell responses begin to 25. Hyslop, P. A., Oades, Z. G., Jesaitis, A. J., Painter, R. G., Cochrane, C. G.,
and Sklar, L. A. (1984) FEBS Lett. 166,165-169
decay. The apparent lifetime of the activated receptor,based 26. Simchowitz, L., Spilberg, I., and deWeer, P. (1982) J. Gen. Physiol. 79,
on cell function, could be a few seconds orless. We have also 453-479
27. Yuli, I., and Snyderman, R. (1983) Clin. Res. 31,379a
generated a model of ligand-receptor dynamics, consistent 28. Zigmond, S. H., and Sullivan, S. J. (1979) J. Cell Biol. 82, 517-527
with high resolution binding data, in which there is a rapid 29. Gerisch, G., and Keller, H.U. (1981) J. Cell. Sci. 52, 1-10
conversion of occupied receptor from a low affinity activated 30. Sklar, L. A,, Omann, G . M., and Painter, R.G. (1985) Fed. Proc. 44,976a;
(1985) J. Cell Biol. 101, in press
to a high affinity inactivated state with a half-time for con- 31. Lefiowitz. R. J.. Caron. M. G.. Miahel., T.., and Studel. J. M. (1982) Fed.
Proc. 41, 2664-2669
version of a few seconds (37). The slowly dissociating, inac- 32. DeLisi, C., and Siraganian, R.P. (1979) J. Immunol. 122,2280-2292

tivated form of the receptor appears to become associated 33. Koshland, D. E., Goldbeter, A,, and Stock, J. B. (1982) Science (Wash. D.
C.) 217,220-225
with the cytoskeleton as part of the overall process which 34. Pozzan, T., Lew, D. P., Wollheim, C. B., and Tsien, R. Y. (1983) Science
regulates cell activation and ligand-receptor processing (38). (Wash. D.C.) 221,1413-1415
Sklar, L. A,, and Oades, Z. G. (1985) J . Biol. Chem. 260,11468-11475
In the accompanying paper(35) we examine the implications 35 36 Sitaramayya, A., and Liebman, P. A. (1983) J. Biol. Chem. 258, 12106-
of Ca2+ asa transient signal contributing tocell response. 121011
37. Skiar,-L. A. (1985) Biophys. J . 47,165a
REFERENCES 38. Jesaitis, A. J., Tolley, J. 0.. Painter, R. G., Sklar, L. A,, and Cochrane, C.
G. (1985) J. Cell. Biochem. 27, 241-253
1. Freer, R. J., Day, A. R., Radding, J. A,, Schiffman, E., Aswanikumar, S., 39. Sklar, L. A,, McNeil, V. M., and Finney, D. A. (1985) Mol. Pharrnacol., in
Showell, H. J., and Becker, E. L. (1980) Biochemistry 19,2404-2410 press
2. Freer, R. J., Day, A. R., Muthukumaraswamy, N., Pinon, D., Wu, A.,

SupplementaryMaterial TO K i n e t i c Assays o f NeutrophilActivation.Unlessothewiseindicated a l l assays were

perfanned a t 37 w i t h 2x100 cellslml.lhespectrofluorimetric assays offreeradical
SignalTransduction and Ligand-Receptor Dynamics i n the Human oroduction u s i n 0 cvtochrome C reduction. membrane decmlarizatonusinaDiS-Cd51. and
Neutrophil.Transient Responses and Occupancy-Response b l a r t a r er e l e a s ;i r i n g the substrateMen-acc-Ala-Alb-Pro-Val-anin~thyl c ~ w ; i n . a11
Relationsatthe Formyl PeptideReceptor perfamed i n e i t h e r an SLM 4800 or SLM 8OW spectrofluorometer,aredescribedelswhere
The a l t e r n a t e assay offreeradicalproduction.requiredfortherateanalyses.using
L.A. Sklar, P.A. Hyrlop, 1.G. Oader. A.J. J e s a i t i s , parahydroyphenylaceticacid, i s describedelsewhere(171. B r i e f l y .c e l l s were suspended i n
R.G. Painter. and C . G . Cochrane the presence o f 1.1~44 PHPA. 5 4 u g h 1 superoxide d i s w t a s e . and 54 ugh1horseradish
peroxidase. The pemxidegeneratedoxidizedthe PHPA toafluorescentproduct which was
MATERIALS #NO METHODS e x c i t e d( i nt h ep l a s t i cc u v e t t e 1a t 323 nn w i t h i t s maximm m i s s i o n a t 400 n. The
fluorescence was c a l i b r a t e dw i t h a k n m sample O f hydrogen pemxide. I n f o w l peptide
Neutrohils.Neutrophils R r e obtained from freshhumnblood by t h e method O f Henson stinulatedneutrophilsthe hydrogen pemxide' i s derived iron superoxideanion v i a superoxide
and & and preparedforthesestudies
a s describedpreviously. The bufferfor
5nH KC1. 147nH NaCl. 1 . M KH2PO4, 1 . l M Wa2HW4. 5.5nU
all dislhltase and horse-radishperoxidase(171.

glucose, 1.5nWCaC12, 0 . M HgW4. l+nl ngC12. The assay O f '"cellaggregation" i s perfanned i n therpectrofluorilneter m i t o r i n g t h e
percenttransmittanceofthecell suspension continuously as a functionoftimefollowing
e+ The highaffinityantibodytofluorescein (61 and FLPEP 1141 wereprepared t h e ex.poSure o fc e l l st o FLPEP. The assay for 'shape change' or n d r a n e r u f f l i n g i s
and c a m c e r i z e d as describedpreviously. performed i nt h es p e c t m f l u o r i m t e rm o n i t o r i n gr i g h ta n g l es c a t t e r i n g( 1 8 1 . The assay for
actin polymerization was perfomedaccordingtothecytonetiic method o f Howardand Meyer
K i n e t i c Assays O f NeutrophllLigandReceptor Dynamics. The f l u o r i m e t r i c and Cytometric (191.
assays ofFLYLPasroclationwith I t s receptoraredetailedelsewhem(141.
Receptor Occupancy andTransient PMN Responses 11465
I n t r a c e l l u l a r Ca+*was monitored with t e f l u o r e s c e n t dye q u i " 2 e s s e n t i a l l y as The c o n t r i b u t i o n o f r e c e p t o r occupancy t o t h e magnitudeandtimecourseoftheresponse
describedbyTsien 1201. N e u t r o p h i l s( 5 x l O q l n l I were incubatedwith 2OuM Ovin 2M was evaluated i n pulseexperiments. A typicalvalueof 3*2 percentreceptoroccupmcygives
(Lancashire Synthesis, LTO., Lancashire. U . K . 1 f o r 20 mtnutes a t 37' i n t h e bufferabovebut r i s e t o 50 p e r c e n t m x i m a l dye response(Ffg.4Cl. in C o n t r a s t t o t h e q u i " 2 e l e v a t i o n .t h e
w i t h o u t CaC12 whichminimized c e l la g g r e g a t i o nd u r i n gt h ei n c u b a t i o np e r i o d . The f r e e additionofantibodytofluorescein had l i t t l e impact on the time course of the response if
Quin 2 was r e m v e d b y w s h i n g andresuspendingthe c e l l s i n Ca*' f r e e b u f f e r a t 15'. t h e r e was S u f f i c i e n t occupancy(up t o -20 seconds de ending upon t h e dose Of FLPEP) t o
e l i c i t an Optimalresponse t o t h a t dose ( n o t s h m . g u t see f o r example r e f . 61. T h a t i s ,
Prior to cell activation, the cells
t oe q u i l i b r a t et ot h e i r
these measwemnts wed excitation at
normal i n t r a c e l l u l a r l e v e l a t 37.
wereresuspended i n Fa" c o n t a i n i n g b u f f e r and a l l o w d
340 nn I r i t h a Corning glass UV bandpars3-54
t h e t i n e course o f b i n d i n g appears t o have a m i n i m 1 i m p a c t on t h e d e p o l a r i z a t i o n and i t s
f l l t e r ) .E m i s s i o n was m n i t o r e d t h r w g h a 49CQ1, 3 c a v i t y i n t e r f e r e n c e f i l t e r (Corion.
H o l l i s t e r , MI. Release o f e l a s t a s e and degranulattan. Ye have p r e v i w s l yd e s c r i b e d I continuous,
r e a l - t i m fa n a l y s i so fe l a r t a r er e l e a s eb yn e u t r o p h l ss t i m u l a t e db y FLPEP (121. The l a t e n c y
cU4P l e v e l s i n s t i m l a t e d n e u t r o p h i l s *ere measured w i t h t h e r a d i o i m n o a s s a y k i t fmm oeriod varied from - 3 tn 5 crrnndc well t h o e n t i r e dose range and theresponse was c a n p l e t e
Hew EnglandNuclear(Barton. MI. C e l l s( 5 r 1 0 6 / n l , 2-3 m l s l were e q u i l i b r a t e d a t 37' f o r Led w i t h1u g h 1c y t o c h a l a s i n B . F o r an optimal
1 0 m i n u t e s p r i o r t o exposure t o FLPEP i n s t i r r e d , t h e r m s t a t t e d p l a s t i c c u v e t t e s under c o n c e n t r a t i o n O f FLPEP lr0.3nHl. 20 p e r c e n t or move O f t h e r e c e p t o r s a r e w c u p i e d a t t h e
e s s e n t i a l l yi d e n t i c a lc o n d i t i o n s as theassays above. A f t e rs t i m u l a t i o n .a l i q u o t s (200-300 t i m e when t h e reroonre i s C m o l e t e . S i m i l a r r e s u l t s are obtained when r i a h t a n o l e
u l l Of t h e c e l l suspensionwere w i t h d r a m a t t i m e d i n t e r v a l s a p d t r a n s f e r r e d t o a g l a s s t e s t s c a t t e r i n g i n t h i p r e s e n c e v f ' c y t o c h a l a s i n B i s ~ u s e d a s ~measure a of-deg&&tioi (18)
tubeheated t o 150' i n ab a t ho fb a l l o t i n i beads ( A t l a s Chemical. San Diega CA). The
Sample b o i l e d w i t h i n 2 seconds. i n h i b i t i n g f u r t h e r a l t e r a t i o n of t h e c M P I & l s( 2 1 ) . The The dependence o f t h e e l a s t a s e r e l e a s e on r e c e p t o r occupancy was examined i n t h e p u l s e
a n a l y s i s f r o m t h i s p o i n t On f o l l o w e d t h e L t h o d o f S i m h o r i t z . e t a l . (22). Because
the protocol(121. These datahave noy been i n c o r p o r a t e d i n t o an OCCUPancy responsecurveshorn
c M P l e v e l i n each m p l e i s l o . r e q u i r i n g t h e m ~ see n s i t i v e a c e t y l a t e d assay and
r i n F i g . 5. He Observe t h a t 50 percentOptimalresponse i s Obtainedfollowingroughly 3
r e c l u d i n g t h e use o f t h e t r l t i a t e d E M P recover marker we i n t r o d u c e dt r a c el e v e l s of percentoccupancy.
%t' into t h ec e l l suspension. T k r e c o Of %kri n :he a l i g y o t st a k e nf o r assay,
after correction for crossover 1%
into t h e channel. was f a c t o r e d i n t o the = w P Freeradicdlgenerationbystiwlatedneutrophils.Neutrophilsgeneratesuperoxide
deteralnation. a n i o nr a p i d l yf o r 2 t o 3 m i n u t e sf o l l o w i n gt h e i re x p o s u r et o FLPEP. Yhen t h e k i n e t i c s o f
t h e responsesare r e p l o t t e d as t h e f i r s t d e r i v a t i v e . or t h e r a t e o f f r e e r a d i c a l p r o d u c t i o n
Pulse f o m t o f n e u t r o p h i ls t i m l a t i o n . In a l lo ft h ek i n e t i c assays c i t e d above. a VI. time. we Obsewe t h a t t h e resoonsesare i n i t i a t e d w i t h i n 10 seconds w h i l e t h e maximal
pulse formt of stimulation is achieved by a h i n i s t r a t i o n O f FLPEP ( < 1 M I i n a S t i r r e d , rate o f p r o d u c t i o n i s achieved w i i h i n -30-60 seconds(171. The t i m ec i u r s e so f theise
t h e r m s t a t t e dp l a s t i cc u v e t t e . The m i x i n gt i m e I n t h i s system i s l e s s t h a n 1 second. I l f t e r responses ave notvery much affectedbythe FLPEP c o n c e n t r a t i o n (EO% -0.3 MI. n b x i m l
a tied Interval. antibody to fluorescein 125 nul i s added t o b l o c k t h e f u r t h e r i n t e r a c t i o n rates o f responsearedetected above 1 nM a f t e r 30 seconds o f b i n d i n g ( 9 0 percent receDtor
o ff r e el i g a n du i t ht h er e c e p t o r . The i n t e r a c t i o n o f t h e a n t i b o d y Ir m id(lessthan 1 occupancy),butthereroonsesarecompletedonlyafterreveralminutes(>70oercent
second)and s p e c i f i c f o r t h e f r e e l i g a n d (6,141. As notedpreviously (6!. n e i t h e r t h e occupancy).
antibodyalone nor t h e c m p l e x of the antibody and f r e e l i g a n d a m m a r k e d l y s t i m u l a t o r y t o
thecells. MOarmver. thepresence o f n e i t h e r t h e a n t i b o d y nor i t s c a p l e r a f f e c t t h e The f r e e r a d i c a l p r o d u c t i o n a f t e r p u l s e s t i m l a t i o n has been examined i n o r d e r t o
subsequent s t i m l a t i o n , o f c e l l s bynon-fluorerceinatedformylpeptide. Yhen antibody i s evaluatethe rimer o f r e c e p t o r s w h i c h a c t u a l l y c o n t r i b u t e d t o t h e s e c e l l r e s p o n s e s . The
present i n t h e c e l l suspension a t t h e t i m e of S t i m u l a t i o n t h e subsequent c e l l responses t o a pulseresponsesare shown i n Fig. 6A and f i r s t d e r i v a t l v e p l o t s a r e shown i n F i g . 68.
mldran e FLPEP c o n c e n t r a t i o n 10.1-0.5 nM dependingupontheresponsebeingmeasured) are F o l l o w i n g a p u l s e o f a h a l f - o p t i m a l dose o f p e p t f d e (-0.3nMl. u e o b r e r r e : 1 ) n a r i n a l r a t e s
t y p i c a ? l y r e d u c e d t o a level corresponding t o a FLPEP c o n c e n t r a t l o n < l o p e r c e n t o f t h e ofresponses are established by FLPEP bindingwhich occurs w i t h i n t h e f i r s t M seconds o f
a r f n i n i s t e r e d dose I i . e . . 0.01 t o 0.05 "MI. Thisresponsereflects FLPEP b i n d i n g t o i t s b i n d i n g ; 2 ) the responses continue for less than 10 seconds a t t h e i r p r e - a n t i b o d y r a t e t h e n
r e c e p t o r sd u r i n gt h et i m e( 1 second) it t a k e r f o r t h e a n t i b o d y t o b i n d t h e f r e e FLPEP. rapidlyfall-offafterantibodyis added; 31 t h e h a l f - t i m e f o r t u r n - o f f Of t h e r a d i c a l
S p e c i f i c d e t a i l s are p r o v i d e d i n t h e d e s c r i p t i o n s O f theindividualresponses(seeResults). p r o d u c t i o n i s a b w t 1 0 Seconds: 41 t h e r a d i c a l p r o d u c t i o n c a n b e i n h i b i t e d b y a n t i b o d y a t
timesof one minuteandbeyond. Yhen Such pulsedataareevaluated i n occupancy-response
C a l c u l a t i o n o f Responses. The e x t e n t of c e l l u l a r r e s p o n s i v e n e s s VaTies w i d e l y w i t h terms we Dbsewe t h a t -30 p e r c e n t o f t h e i e c e D t o r s g i v e r i s e t o -50Dercent o f t h e r a d i c a l
c e l l s f r o mi n d i v i d u a l donors. mreover. c e l lr e s p o n s i v e n e s st e n d st od i m i n i s ha s a function p r o d u c t i o n( F i g . 6Cl. A c o n s i d e r a b l y s r n a l 1 e ~ ' f r a c t i o n o f r e c e p t o r s ire r e q u i r e d t o e l i c i t a
oftimeaftercellisolation even though c e l l v i a b i l i t y r m a i n r g r e a t e r t h a n 95 percent half-optimal rate of response (i.e.. 15-20 seconds O f b i n d i n g a t 0.3 nM or about7-10
(231. In most experiments h e r e p u l s e r t i m l a t i o n was a h i n i s r e r e d , experimentsfollowed percent of the receptors).
t h i s p r o t o c o l : w i t h i n ?he f i r s t 2 hours f o l l a i n g i s o l a t i o n o f t h e c e l l s , dare-response
curves w v e determined;subsequentlyaulse-responseanalysis f o r p a r t i c u l a r FLPEP C e l la g g r e g a t i o n .N e u t r o p h i l sa g g r e g a t er e v e r s i b l y( i nt h e absence o fC y t o c h a l s i n BI
concentrationsle.g. 0.1. 0.3, o r 1.6 My ws obtained, The response O f t h e c e l l s a t t h a t over a period of a feu minutes. as m n i t o r e d by the change i n transmittance of a c e l l
tie i n t h e absence g f t h e a n t i b o g was r e d e t e m i n e d . The fractionalresponsestowardthe suspensionfollowingexposureto FLPEP ( F i g . 7Al. Ye haveexamlntdtransmittance i n pulse
Pulse were normalized t e t h e r e r p o n r e o b t a i n e d f o r t h e same dose a t t h a t time.Typlcally. experiments under conditions parallel t o those described for each O f the other responses.
l e s s t h a n 30 p e r c e n t r e r p n s i v e n e s r was l o s t between t h e t i m e o f t h e dose-response and t h e Nearoptirnalaggregation i s detected i n the same dose range (above 0.3 nH FLPEPl a s
pulse-response. superoxidegeneration, Uhen b i n d i n g i s i n t e r r u p t e d byaddingantibody t o fluorescein.the
m nitudeoftheresponse can bediminishedaftertimes d l l o n g as 60 seconds or more ( F i g .
RESULTS 7B!. Assuming thattheaggregation i s p r o p o r t i o n a lt ot h ei n c r e a s e i n transmittance. it Is
apparentthatthe O C C U P ~ C Yrelationship is Similar to that Observed f o r superoxide
Receptoroccupancyunder pulseconditions. The dynamics o f i n t e r a c t i o n o f FLPEP w i t h production. The l a t e n c y for recovery (or r e v e r r i b i l i t y ) a f t e r a n t i b o d y a d d i t i o n i s trenty
i t s r e c e p t o r s have been c h a r a c t e r i z e di nar e c e n tp u b l i c a t i o n( 1 4 ) . The f r a c t i o n a l receptor
Occupancy based On t y p i c a l r a t e c o n s t a n t s f o r t h e c e l l
thetimesrelevant io
~ . . ..
s t i m l a t i o n a m s u m r i Z e d ~ i n - T a b l e S-I. Formidrangepeptide
t h e FI DFP dnrar ",,"
""~ =-A
t o t h i r t y seconds.DisaggregationfollowingpulseStinulation
a f t e r whichthe Cat+ signal has alreadyapproached i t s basallevel.
i s detectedonly a t timer

c o n c e n t r a t i o n (0.1 t o 0.3 nM) -4 t o 1 3 p e r c e n t o f t h e r e c e p t o r s a r e o c c u p i e d a f t e r 30 T r a n s l e n tr l g h t angle l i g h t s c a t t e r W S ~ O ~ and S ~ actinpolymerization. I n t h e absence

secondsof b i n d i n g a t 37'. B i n d i n ga tl o w e r doses and shortertimes i s n e a r l y l i n e a r . Of c y t a c h a l a r l n B, neutrophlsundergo d t r a n s i e n tr e d u c t i o n 1-15 p e r c e n t ) i n r i g h t anqle
In F i g u r e 1 , typicalbindingdataObtainedunderpulseconditions are displayed.There s c a t t e r i n gs h o r e m a x i m m occurs M i t h i n 8-17 seconds 118.77). Y u l l and Snydernan (77)have
are twoelements o f thebindingdatacentralto'pulse"responseexperimfnts:1)thebulkof p ~ o v t d e devidence t h a t t h l s responserepresentstherapidmmbraneruffling (28.29j detected
theliganddissociationproceeds with an apparent half-time Of -3-5minutes; 2) when c e l l s are exposed t of o r m y lp e p t l d e . The response 7s near optimal above .01 nM FLPEP
internalization of the ligand has an apparentlatencyof -30 secondsandhas a half-timeof ( 1 8 ) . I n t e n Seconds ofblnding O f 0.02 nu, -0.2 Percent ( o r -20 o f 60,a)O receptors)of
-3 minuter114).There two f e a t u r e si n d i c a t et h a td u r i n gp u l s ee x p e r i m e n t so f two minutes t h er e c e p t o r s are Occupied. In p u l s e e r p e r i m n t r Only 7-5 recondr Of b i n d i n g , an estimated
b i n d i n g o r l e s s , mrt o f t h e r e c e p t o r r m a i n s bound t o t h e membrane during the time i n which 20 r e c e p t o r r l c e l l are r e q u i r e dt oi n i t i a t e near o p t i m a lr u f f l i n g ( F l g 8 ) . When b i n d i n gi s
c e l l responses are e l i c i t e d and t u r no f f . lnhlbitedhoiQver,the recovery o f t h e I l g h t s c a t t e r r i q n a l towardbaselineproceedswith a
h a l f - t l m o f 10-15 seconds.
The measuremfnt o f i n t r a c e l l u l a r Ca'+ responseswiththefluorescent d e q u i n 2. The
e l e v a t i o n 07 m e tluorescence O f i n t r a c e l l u l a r Q u i n 2 i n response t o F L k i o n i s
shown i n F i g . 2A. Optimalfluorescenceresponsesweretypicallyobserved a t doses above 0.3
nH FLPEP whilehalf-optimalfluorescenceresponses were Obtained a t -0.05 nM FLPEP. For
Optimaldosestheresponseswere i n i t i a t e d i n t h e f i r s t o r secondtimeintervalirecondsl
f o l l o r i n g s t i m u l a t i o n and maximal fluorescence was Observed i n 3-5 seconds. F o r l a e r FLPE
concentrations.thelatencyperiodsincreasedtotimes up t o 5 seconds and t h e t i m e t o
maximalresponseincreased t o times as l o n g a s 10 seconds.

The r e l a t i o n o f Ca" m o b i l i z a t i o n t o r e c e p t o r occupancy was examined under p u l s e

c o n d i t i o n s( F i g 201. Nearoptimalresponsesare obtained when pulses a w longerthan 7
nu. Such apulseoccupies '1 percent O f thereceptors.
added a t any time during-the time courseof binding (the
half-timefarbindingfor 0.2 nM FLPEP i s -1 minute a t 37 ) , b u t f o l l a i n g t h e mdximal
responsetheQui" 2 levelsfalltowardrestinglevelswith a h a l f - t i m e O f -10-15 seconds.
S i m i l a r r e s u l t s are Obtained a t FLPEP c o n c e n t r a t i o n s up t o a t l e a s t 1 nM and b i n d i n g t i m e r
o f 1 min ( a t which time a t l e a s t 50 percentofthereceptors may beoccupied).Antibodyhas
no e f f e c t on t h eS t i m u l a t i o n O f c e l l s bythenon-fluorescentpeptide(notsh-I. Thus.
i n t r a c e l l u l a r Cat' l e v e l s may be mdxirnallyelevatedbyverylowlevels of receptor
o c c u p a ~ c y ; however. Caf* l e v e l s are r a p i d l y r e t u r n e d t o r e s t i n g i e v e l f unless FLPEP
b i n d i n gp r o c e e d st h r o u g h w ti t s normaltime course.
cWP. The i n t r a c e l l u l a r l e v e l s o f cAHP r i s e t r a n s i e n t l y t o maximal l e v e l s w i t h i n 10.20
secoFZ-and r e t u r nt ob a s a ll e v e l sf o l l a i n g 90 t o 120seconds 122, 24, 251. There i s
i n s u f f i c i e n t r e s o l u t i o n i n thedatatodeterminethelatencyperiod. The dose response
Curves show h a l f - m r i m a l r e s p o n s e s a t -0.1 nH FLPEP and maximalresponsesabove 0.3 nn [ F i g .
3Al.Consideringthat -7 percent of t h er e c e p t o r s are occupied i n 15 recands a t 0.3 nu
FLPEP (Table S-11, only a smallfTaction o ft h er e c e p t o r sa p p e a rt op a r t i c i p a t e in
i n i t i a t i n g cAHP e l e v a t i o n .
Thisissue was c l a r i f i e d i n p u l s e e i p e r i n e n t r ( F i g . 381. Doses of FLPEP (0.1 1 nM) -
uere p u l s e d f o r p e r i o d s up t o IO seconds.Forexample, a 10 second, 0.3 nM p u l s e was
s u f f i c i e n tt oe l l c i tn e a ro p t i m ll 7 9 * 2 9p e r c e n t )c e l l responses. He observe t h a t 50
percent maximal responsearises frm 1-2percentreceptoroccupmcy.There i s insufficient
r e s o l u t i o n i n W P k i n e t i c d a t a t o determinewhetherthetime course O f cWP e l e v a t i o n was
influenced by the p u l s e p r o t o c o l .

De o l a r i z a t i o n . The fluorescenceresponsesofthe d r a m p o t e n t i a ls e n s i t i v e dye 06 12 18 24 30

OiS-*ved t o r e f l e c t a mmbrane d e p o l a r i z a t i o n i n t h e n e u t r o p h i l causedby {he 1
.. ,-,-I
i n f l u x O f Na' and e f f l u x O f K'. S i m h o w i t za n d cawrkers 1261 have providedevidence
t h a t the change i n themmbranepotential i n t h en e u t r o p h i l i s r w g h l y l i n e a r w i t h t h e Fiqure1 The B i n d i n g and O i l s o c i a t i o n Of FLPEP Under Pul~e-TypeConditionr.
change i n f l u o r e s c e n c e i n t e n s i t y o f t h e dye. The t i m ec o u m e anddose-responseofthe
DiS-C35 response d ~ eshown i n Figs. 4A and 48. The response k i n e t i c s are m i l d l y A ) The d a t a are p l a t t e d as thefluorescence of FLPEP v s . time. The method was
dependent upon t h e FLPEP c o n c e n t r a t i o n w i t h l a t e n c y p e r i o d s o f - 5 t o 9 seconds w e ? a 1w errentially as described ( 1 4 ) w i t hm d i f l c a t i o nd e s c r i b e d below. A c e l l suspension(107
f o l dc o n c e n t r a t i o n rangespanning Kd. The maxim1wsponse
40 second^ a f t e r s t i m u l a t i o n and recoveryrequires
i s d e t e c t e df o l l o w i n g -25 t o
2 t o 3 minutes.For a near optimal FLPEP
R I N l n l l was
ewosed to 1 nM FLPEP. A f t e r 15, 30.
#as added t o b i n d and quench f r e el i g a n d .
60.or 120 Secl. 2 5 nM antibody t o
The c u w c s representthemount bound
c o n c e n t r a t i o n ( 1 "ti). mre than 30 percent of the wceptovs are occupied a t t h e t i m e o f t h e a t t h e ti= o f a n t i b o d y a d d i t i o n and t h e d i s s o c i a t i o n O f l i g m d frm thereceptorthereafter
Optimalresponse. canpared t o suspensions i n whichbindinghad been blockedbythepresence O f 10%
t8oc-phe-leu-phe-leu-phe. T r , m d i f i c a t i a n si m p r o v i n g an r e f . 14 wew enplayed i n these
b i n d i n g assays: 1)superoxidedismtase ( 8 u g l m l ) and bovinecatalase ( 8 uglml) were
Included i n order t o p r e v e n t o x i d a n t danage t o t h e C h P m p h o r e Of FLPEP. 2 ) 15 nw NHq i s
Dccarronally. we observed thatweparationsofantibody or BSA i n whichpeptide was lncluded i n thebuffertomaintainthefluorescence Of i n t r a c e l l u l a r FLiEP, a g a i n s t
d i s s o l v e d were s l i g h t l y s t i m u l a t o r y i n t h e Ouin 2 d e t e m i n a t i m s . T h i s a c t i v i t y lvsoraaaldcidificatim. These m o d i f i c a t i o n s a l l o w us t o m a i n t a i n >95 Dercent O f t h e FLPEP
apparentlyrepresentedsmallquantities O f aggwgatedproteinwhichCould be removed b? fiuarescence.
c e n t r i f u g a t i o n .Y h i l et h ea g g r e g a t e - f r e ea n t i b o d yt of l u o r e s c e i ni t s e l f was
" o n - ~ t i m l d t o r y , t h e preformedcomplex of antibody and FLPEP produced a verylowlevel B) The S p e c i f i cb i n d i n g and d i s s o c i a t i o n 1i.e.. 'bound minusblacked') Of FLPEP VI.
responre.probably due t o d i s s o c i a t i o n O f FLPEP upon i t s d i l u t i o n . time a f t e r 15. M. 60 o r 120 r e c m d r o f b i n d i n g .
11466 Receptor
Occupancy Transient
and PMN Responses

FLPEPinMl TimeiSeondsl

+ Anti
+ FL

z 01 1 2 1

L t ~ ~ ~ ~ ~ ' l ' 1
0 20 40 60
Time (Seconds\
F i g u r e 2 The K i n e t l c s ( A ) and Pulse-Response ( B ) ofQuin 2 i nF o m y lP e p t i d eS t i m u l a t e d

l o oyT~'
A) The data are p l o t t e d i n terms o f t h e p e r c e n t maximal fluolercence response VI time
where thestimulus I S thefluOreSceindtedherapeptide. BSn i s t h e c o n t r o l l i t h w t FLPEP.

8 ) The response i s e x p l e ~ ~ eads t h e percent maximalfluorescenceresponse O f @ i n 2 VI

time. The Stimulus i s 0.2 nH fluoresceinatedherapeptidefollowed a t 10. 30. o r 50 seconds Fractionll Ascsptor Occupancv
by t h ea n t i b o d y t o fluorescein.AntiFLreprerentstheantibodyalone.Cmplex i s the
preformedcmplerofAntiFL and FLPEP. -4 Response o f OiS-C2(5) i nS t i w l a t e dN e u t r o p h i l s .

A ] The k l n e t ? c r o f t h e dye response are shown VI theConcentration O f the

fluorerc?lnatedpeptlde. he dataisexpressed as the r e l a t i v ef l u o r e s c e n c ei n t e n s i t y YS
80 8 ) ~ o s e - r ~ ~ p a ncurves
se o f dye response YS s t l n u l u sc o n c e n t r a t i o n . Oata f o rn e u t r o p h i l s
frm 5 1 % donors I s r h a n .

c ) occupancy-responsecurves f o r t h e dye-response. Pulse FeSponSe e x p e r i w n t r *ere

60 ~ ~ n d ~ for e d donors and the d a t a are expressed as thepercent maximalfluorescence
~ t s~~
131 response V I t h ec a l c u l a t e df f a c t l o n a lr e c e p t o r occupancy.

c 20
Y 0.5 10 3.0 10.0 0

% Receptor Occupancy
Elastase Release Pulse-Response.

The d a t a are expressed as t h e p e r c e n t m x I n a 1 e l a s t a s e r e l e 8 s e V I t h ep e r c e n tr e c e p t o r

t h e s i ' e r & i " P n t rc e l l s were s t i m u l a t e d $ n t r i p l i c a t e and a l i q u o t l were t a k e n a t t h e occupancy. m e s o l i d spbols ( a ) r e f l e c t d a t a s u m r l z e d fmn 4 donors w i t h t y p i c a l error
p l a t e a uo ft h er e i p o n r e( 1 0 20 and 30 seconds). The data frm a11 t h e s et i m ep o i n t s ere bars (see Ref. 121. The t r i a n g l e s r e f l e c t e l a s t a s e r e l e a s e i n a s i l l l l t a n e o u s a n a l y s i s Of
averaaed. The nmber ~n pa;ent/lenlrepresentsthe number o f I n d i v i d u a l donors for which
degranulation(Ref. 181. The open c i r c l e s r e f l e c t a s i x t h donor.
and Transient PMN Responses 11467

0.3nM FLPEP TidSscmdSl FLPEP TimdSecondsl

Xna ISscondrl
The Transmitted Light ("Aggreqation'l Response of N e u t r o p h i l s .

A) Adose-responseanalysis of t h ek i n e t i c s O f aggPegation i s shown. The data are

loo- p l o t t e d 13 t h e r e l a t i v e l i g h t t r a n s m i s s i o n V I tim. The s t i m u l u s M S t h ef l u o r e r c e i n a t e d
c A peptide.
8) The pulseresponseofthekineticsofaggregation i s shorn. The S t i m l U S was 1 nt4
fluorerceinatedpeptide. The pulseswere 0. 10, a, or 60 seconds. S i m i l a r reruitr are
o b t a i n e d f o r 0.3 nM peptide ( n o t shown).

Percent Receptor Occupancy

m 6 Pulse-ResponseAnalysis of F r e eR a d i c a lP r o d u ~ t i o n

AI The response t o p u l s es t i m l u a t l o n O f n e u t r o p h i l s i n t h e PHPA assay i s r h a n . The

pu11c1 are 0. 10. 30.
C a l i b r a t e dN i t hr e s p e c tt o
or 5U seconds. The d a t a i s s h o r n as t h e PHPA fluarescence YS time.
O i e q u i v a l e n t s .S i n l l a rr e r u l t s are obtained *hen t h e
c y t o c h r m C assay i s used l R e f 6).

B l The f i r s t d e r i v a t i v e of t h e d a t a from A are p l o t t e d . The dataareexpressed as the

p e r c e n t o f t h e m x l m a l rate o f p r o d u c t i o n (cmpared t o t h e c o n t r o l w i t h o u t a n t i b o d y ) YS

Cl An occupancy-responsecuwe i sc o n s t r u c t e d from pulseexperiments. The open s y m o l s

r e p r e s e n t f o u r separate e x p e r i m e n t s i n v o l v i n g c e l l s f r m t h r e e d i f f e r e n t donors and t h e
cytochrome C assay. The f i l l e d syubols a t e representativedata from l e s se x t e n s i v e
experimentsusingthe PHPA assay. The data a r e p l o t t e d a s thepercent maximal f r e e r a d i c a l
generation VI t h ec a l c u l a t e df r a c t i o n a l receptor occupancy.

Receptor Occupancy VI
Time and Ligand Concentration
200 1
for P u l s e S t i m l a t i o n a

T i mo f Antib0d.y Length o f Bindingb

Receptor Occupancy
A d d i t l oPn e( rrei ocdl (secl Ligand Concentration Inn)
0.01 0.03 0.1 0.3 1.0

0 0.017 0.05 0.17 0.5 1.7

2 0.05 0.15 0.5 1.5 4.8
5 0.10 0.29 1.0 2.9 9.3
15 0.23 0.71 2.3 6.9 21
M 0.43 1.3 4.4 13 36
60 0.80 2.4 1.8 21 55
0 60
1 120 BO
a. The b l n d i n u datA a mC I l C u l a t e da c c o r d l n a t o Ref 39 u s i n 0t h er a t e
Time lrecondrl
Fiqure 9 The F V t i n P o l y w n r a t i o n Response Of N e u t r o p h i l s t o Pulse S t i m l a t i o n .
b. The p e r i o d of b i n d i n g i s e l t i s a t e d frm t h e O b s e w a t i o n t h a t t h e The data are p l o t t e d as t h er e l a t i v eF - a c t i nc o n t e n tp e rc e l l VI. time. The curves
h a l f - t i n e for i n t e r a c t i o n between t h e a n t i b o d y t o f l u o r e s c e i n and FLPEP represent responses t o 1 nM FLPEP ( e ) w i t h m t i b c d y added after 15 ( a ) . 60 ( 0 ) . or 180 ( A )
i s -1 sec (61. FOT t h e values i n b r a c k e t s c a l c u l a t i o n s are based on 15.
30. M) * e c * rerpectire1y.