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Desensitization of Formyl Peptide Receptors Is
Abolished in Calcium Ionophore-Primed
Neutrophils: An Association of the
Ligand-Receptor Complex to the Cytoskeleton Is
Not Required for a Rapid Termination of the
NADPH-Oxidase Response
Li Liu, Olle Harbecke, Hans Elwing, Per Follin, Anna Karlsson
and Claes Dahlgren
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 1998 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 1998; 160:2463-2468; ;
http://www.jimmunol.org/content/160/5/2463
Desensitization of Formyl Peptide Receptors Is Abolished in
Calcium Ionophore-Primed Neutrophils: An Association of the
Ligand-Receptor Complex to the Cytoskeleton Is Not Required
for a Rapid Termination of the NADPH-Oxidase Response1
Li Liu,2* Olle Harbecke,* Hans Elwing,† Per Follin,‡ Anna Karlsson,* and Claes Dahlgren*
T
he neutrophil-mediated defense against microbial infections is dependent on a unique ability of the phagocytes to
produce large amounts of reactive oxygen species, i.e.,
superoxide anion and hydrogen peroxide (1, 2). These oxygen radicals are generated by an enzyme system, the NADPH-oxidase,
which is assembled upon activation of the cells (3, 4). The
NADPH-oxidase is triggered by many different stimuli, among
which the chemotactic peptide FMLP is a prominent example (5,
6). Most neutrophil receptors (including the receptors for FMLP)
are stored in subcellular organelles (for review, see Ref. 7), and in
order to respond properly to various stimuli, the neutrophils have
to recruit the different intracellular granule subsets to the plasma
membrane, mobilizing new receptors to the cell surface (8). This
process also endows the neutrophil plasma membrane with increasing amounts of the membrane-spanning component (the b
cytochrome) of the NADPH-oxidase (7, 9).
Binding of FMLP to the neutrophil FMLP receptor (FMLP-R)
activates several signal transduction pathways (5, 10, 11). However, the precise signal(s) responsible for the subsequent activation
*The Phagocyte Research Laboratory, Department of Medical Microbiology and Immunology, †Department of General and Marine Microbiology, University of Göteborg, Göteborg, Sweden; and ‡Department of Infectious Diseases, University of
Linköping, Linköping, Sweden
Received for publication December 5, 1996. Accepted for publication October
30, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by The Swedish Medical Research Council, The King
Gustaf V Memorial Foundation, The Swedish Association Against Rheumatism, The
Anna-Greta Crafoord Foundation, The Fredrik and Ingrid Thuring Foundation, and
The Anna-Brita och Arne Lundberg Foundation.
2
Address correspondence and reprint requests to Dr. Li Liu, The Phagocyte Research
Laboratory, Department of Medical Microbiology and Immunology, University of
Göteborg, Guldhedsgatan 10, S-413 46 Göteborg, Sweden.
Copyright © 1998 by The American Association of Immunologists
of the NADPH-oxidase has not yet been identified. The amount of
superoxide anion and hydrogen peroxide released upon neutrophil
activation with FMLP is influenced not only by the degree of receptor exposure on the cell surface (12, 13) but also by the termination of the oxidase activity. The signals that lead to termination
have not yet been defined. It has been suggested that the ability of
the receptor-ligand complex to generate transmembrane signals is
lost when the complex associates with the cytoskeleton, an event
known to follow shortly after the binding of FMLP to its receptor.
It has been convincingly shown that this cytoskeletal binding leads
to desensitization of the receptor with respect to further activation
by the agonist (14 –17). It has also been suggested that the coupling
of the receptor-ligand complex to the cytoskeleton is responsible
for the termination of an ongoing FMLP-induced NADPH-oxidase
response (14 –16).
It is well known that neutrophils that have mobilized their intracellular FMLP-R stores are “primed” with respect to FMLPinduced NADPH-oxidase activation. One mechanism behind this
state must be an increased generation of second messengers that
activate the NADPH-oxidase, but other regulatory mechanisms
might also be of importance. The present study was performed to
reveal whether priming following a rise in intracellular calcium is
associated with a change in cytoskeleton-mediated regulation of
FMLP receptor activity. The fact that desensitization was abolished in calcium ionophore-primed cells also made it possible to
investigate the role of desensitization for the termination of the
oxidative response.
Materials and Methods
Reagents
Dextran and Ficoll-Pacque were purchased from Pharmacia (Uppsala,
Sweden). FMLP, cytochalasin B, isoluminol, and Triton X-100 were obtained from Sigma (St. Louis, MO). Ionomycin was purchased from Calbiochem (La Jolla, CA). The phycoerythrin-conjugated mAb against CR3
0022-1767/98/$02.00
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Binding of ligands to N-formyl peptide chemoattractant receptors exposed on human neutrophils generates signals in the cells that
induce an activation of the superoxide anion producing NADPH-oxidase. Ligand binding is followed by a rapid association of the
ligand-receptor complex with the cytoskeleton, a process leading to desensitization of the cells with respect to NADPH-oxidase
activation. We show that neutrophils that have experienced an intracellular calcium rise obtained through interaction with the
calcium-specific ionophore ionomycin are “primed” with respect to the FMLP-induced production of superoxide anions. Mobilization of FMLP receptors from intracellular pools is one well-known mechanism behind the primed response. Based on our
finding that ionomycin-treated neutrophils could not be desensitized, we suggest that the lack of association between the ligandreceptor complex and the cytoskeleton is an additional priming mechanism. Since in vivo-exudated neutrophils, which also had
mobilized intracellular organelles, could be desensitized, we suggest that the abolished desensitization in ionomycin-treated neutrophils is not due to an inability of newly recruited receptors to couple to the cytoskeleton. We show that a rapid termination of
FMLP-induced superoxide anion production is obtained in both desensitizable and nondesensitizable neutrophils, suggesting that
the desensitization phenomenon is of limited importance in the oxidase termination process. The Journal of Immunology, 1998,
160: 2463–2468.
2464
LACK OF FMLP-R DESENSITIZATION IN IONOMYCIN-TREATED NEUTROPHILS
was purchased from Becton Dickinson (San Jose, CA). The radiolabeled
peptide [3H]FMLP was obtained from New England Nuclear (Boston,
MA), and FITC-labeled formyl-Nle-Leu-Phe-Nle-Tyr-Lys (FITC-FN
LPNTL)3 was from Molecular Probes (Eugene, OR).
Isolation of phagocytic cells
Blood neutrophils were isolated from heparinized whole blood or from
buffy coats obtained from apparently healthy adults. After dextran sedimentation at 1 3 g, hypotonic lysis of the remaining erythrocytes, and
centrifugation in a Ficoll-Paque gradient (18), the neutrophils were washed
twice and resuspended in Krebs-Ringer phosphate buffer containing glucose (10 mM), Ca21 (1 mM), and Mg21 (1.5 mM) (KRG, pH 7.3).
Exudated neutrophils were harvested from skin chambers placed on
unroofed skin blister lesions on the volar surface of the forearms of healthy
human volunteers, as previously described (19, 20). In each experiment,
two chambers with three 0.6-ml wells covering the lesions were used. The
chambers were filled with autologous serum, and the neutrophils were allowed to accumulate in the chambers for 24 h. More than 95% of the cells
harvested from the chambers were neutrophils.
Measurement of neutrophil superoxide anion production
Mobilization of subcellular organelles
Neutrophil subcellular organelles were mobilized by treating the cells with
ionomycin (22), a calcium-specific ionophore. After preincubating the cells
for 5 min at 37°C, ionomycin (5 3 1027 M final concentration) was added,
and the incubation was continued for 5 min. The cells were then centrifuged, washed once, resuspended in KRG, and put on ice until used.
Desensitization
Neutrophils (107/ml) were incubated for 5 min at 15°C, FMLP (1027 M
final concentration) was added, and the incubation was continued for 10
min. The cells were then added (50 –100 ml) to prewarmed (37°C) CL vials
containing isoluminol, HRP, and FMLP (1027 M final concentration), and
the production of superoxide anion was followed as described above.
Determinations of receptor exposure by FACS analysis
Neutrophils were fixed for 30 min in ice-cold paraformaldehyde (4% w/v
in PBS).
To determine the exposure of CR3, 10 ml of a conjugated mAb was
added to a cell pellet (;100 ml) of 106 cells. The cells were incubated on
ice for 30 min, washed twice with KRG, and analyzed for amount of cell
bound probe (correlating to the amount of CR3) by flow cytometry (FAC
Scan; Becton Dickinson, Mountain View, CA).
To determine the exposure of FMLP-R, a FITC-conjugated formylated
peptide (FITC-FNLPNTL; 1028 M final concentration) was added to a cell
pellet (;100 ml) of 106 paraformaldehyde-fixed cells in the absence or
presence of an excess amount (5 3 1026 M) of nonlabeled FMLP. The
cells were incubated at 22°C for 30 min, and no washing was performed
after labeling. The amount of specifically bound probe (correlating to the
amount of FMLP-R) was determined by flow cytometry (23).
The cellular content of F-actin
The F-actin content in the neutrophils was analyzed by staining with fluorescein-phalloidin (Molecular Probes). The cells were fixed with paraformaldehyde (4% w/v in PBS), permeabilized with phosphatidylcholine, and
3
Abbreviations used in this paper: FNLPNTL, formyl-Nle-Leu-Phe-Nle-Tyr-Lys;
HRP, horseradish peroxidase; CL, chemiluminescence; SOD, superoxide dismutase;
KRG, Krebs-Ringer phosphate buffer containing glucose, Ca21, and Mg21; Mcpm,
106 counts per minute.
FIGURE 1. Time course of the neutrophil superoxide anion production
following activation with FMLP (1027 M final concentration) measured by
isoluminol/HRP-amplified CL. The curves are from a representative experiment and show the response induced in cells pretreated with ionomycin
(5 3 1027 M, 5 min; dashed line) before challenge with the peptide and in
control cells (incubated in the same way but without addition of the ionophore; solid line). FMLP was added at time 0. Abscissa, time of study
(minutes); ordinate, superoxide anion production (Mcpm).
incubated with FITC-phalloidin according to the instructions of the manufacturer. The cell content of F-actin was determined by flow cytometry.
Association of the ligand-receptor complex with the cytoskeleton
To determine the amount of receptor-ligand complex associated with the
cytoskeleton, the cells were allowed to interact with FMLP at 15°C. Interaction with the peptide at 15°C permits binding of the ligand-receptor
complexes to the cytoskeleton, but neither the mobilization of intracellular
organelles nor the internalization of the receptor-ligand complex occurs
(14, 24). Cytoskeletal fractions were prepared as described earlier (24).
Briefly, the cells were incubated at 15°C for 5 min, after which [3H]FMLP
(2 3 1028 M) was added, either alone or together with an excess of nonradioactive FMLP (1025 M), and the incubation was continued for an
additional 10 min. The cells were pelleted and resuspended in an ice-cold
buffer containing Triton X-100 (1% v/v). The samples were mixed and put
on ice for 10 min, after which they were centrifuged for 20 s (9000 3 g)
in a Beckman microfuge (Beckman Instruments, Fullerton, CA). The Triton X-100-insoluble pellet was washed once in the same medium, and the
radioactivity associated with the cytoskeleton was determined (24). No
radioactivity was associated with the cytoskeleton (Triton X-100-insoluble
pellet) when the peptide was introduced in the system 30 s after solubilization with Triton X-100.
Results
Superoxide anion production and mobilization of neutrophil
granules
The chemoattractant peptide FMLP induced a very rapid neutrophil response measured as superoxide anion production (Fig. 1).
The maximal production was reached within the first minute, and
the response was terminated within 3 to 4 min.
The major part of the neutrophil FMLP-R is stored in subcellular vesicles (secretory vesicles) and mobilizable granules (gelatinase granules and specific granules; for review, see Ref. 7). The
mobilization of these intracellular organelles, following an intracellular Ca21 rise (obtained by ionomycin treatment), subsequently endows the neutrophil plasma membrane with new receptor molecules, as shown for CR3 and the FMLP-R (Fig. 2).
Accordingly, ionomycin-treated neutrophils showed a primed response to FMLP (Fig. 1).
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Neutrophil production of superoxide anion was assayed with an isoluminol-enhanced chemiluminescence (CL) system (21). The CL activity was
measured in a six-channel Biolumat LB 9505 (Berthold Co., Wildbad,
Germany), using disposable 4-ml polypropylene tubes with a reaction mixture of 0.9 ml containing 1 3 106 neutrophils, HRP (4 U), and isoluminol
(a membrane-impermeable CL substrate, 2 3 1025 M). This set-up measures the release of superoxide anion from the cells (21). The tubes were
equilibrated for 5 min at 37°C, after which either the stimulus or, in some
experiments, the cells were added. The light emission was recorded continuously. By a direct comparison of the SOD-inhibitable reduction of cytochrome c (using a millimolar extinction coefficient of 21.1 for cytochrome c (23, 24)) and SOD-inhibitable isoluminol-amplified CL, 7.3 3
107 counts were found to equal the production of 1 nmol of superoxide
anion.
The Journal of Immunology
Ionomycin treatment induced a rapid (maximal within 1 min)
and transient (back to resting value after 5 min) increase in the
cellular content of F-actin, revealed by FITC-phalloidin staining
(Fig. 3).
FIGURE 4. Desensitization of neutrophil superoxide anion production
in control neutrophils. The cells were first incubated at 37°C (to make the
comparison with ionomycin-treated cells valid), washed, and then incubated at 15°C in the presence (dashed line) or absence (solid line) of FMLP
(1027 M). The vials for measuring CL, containing HRP (4 U), isoluminol
(2 3 1025 M), and FMLP (1027 M), but no cells, were warmed to 37°C
in the luminometer. The cells were transferred from 15°C to the measuring
vials (37°C) at time 0, and the light production was recorded. The curves
are from a representative experiment. Abscissa, time of study (minute);
ordinate, superoxide anion production (Mcpm).
Interaction of the receptor-ligand complex with the cytoskeleton
determined as desensitization and Triton X-100 insolubility
Neutrophils that were allowed to interact with the chemoattractant
FMLP at 15°C were desensitized (Fig. 4), i.e., when these cells
were transferred to 37°C, they did not respond to FMLP. This
desensitization was stimulus specific. In fact, neutrophils desensitized to FMLP were primed in response to fluoride ions (presumably acting in conjunction with trace levels of aluminum), while
the PMA-induced response was unchanged (not shown). We as
well as others have shown earlier that binding of FMLP to its
receptor at 15°C is associated neither with any granule secretion
nor with activation of the NADPH-oxidase (24, 25).
In contrast to what was found when using cells with their vesicles/granules intact (control neutrophils), no desensitization could
be induced in ionomycin-treated neutrophils (Fig. 5). This finding
suggests that the Ca21-induced mobilization of vesicle/granulelocalized receptors is associated with an impaired binding between
the occupied receptor and the cytoskeleton. When the level of cytoskeleton coupling of the occupied receptors was determined by
extraction of ionomycin-treated neutrophils with Triton X-100, the
number of cytoskeleton-bound receptors (i.e., receptors detected in
the detergent insoluble fraction) was indeed reduced, despite the
fact that the number of surface-exposed receptors was increased
(Fig. 2).
Binding of FMLP at 15°C was not associated with any measurable increase in the cellular content of F-actin in either of the cell
populations studied (not shown).
Desensitization in exudated neutrophils
FIGURE 3. Effect of ionomycin on the neutrophil content of F-actin.
The neutrophils were stimulated by ionomycin (5 3 1027 M final concentration), and at the times indicated in the figure, samples were taken and the
cellular content of actin was determined and expressed as percentage of the
time 0 value for each individual experiment. The results are given as
mean 6 SD for seven separate experiments.
The lack of desensitization (as well as the impaired anchoring of
FMLP-occupied receptors to the cytoskeleton) in ionomycintreated neutrophils could be explained if the receptors mobilized
from the storage pools lacked the ability to become desensitized.
To test this hypothesis, we determined the ability of exudated neutrophils to become desensitized.
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FIGURE 2. Surface expression and cytoskeleton binding of receptors,
in ionomycin-treated cells (5 3 1027 M; 5 min). The results are expressed
as percentage of the control values obtained when using nontreated control
neutrophils. The cell surface expression of CR3 and receptors for FMLP
was determined by FACS analysis using Abs against CR3 and a FITCconjugated peptide, FNLPNTL, respectively. The Triton X-100-insoluble
(specifically bound) material was used to measure the receptors associated
with the cytoskeleton using a radioactively labeled peptide. The numerals
represent mean values 6 SD of three (CR3 and FMLP-R surface expression) or four (FMLP-R association to the cytoskeleton) independent experiments, respectively.
2465
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LACK OF FMLP-R DESENSITIZATION IN IONOMYCIN-TREATED NEUTROPHILS
FIGURE 6.
Superoxide anion production in exudated neutrophils.
Time course of the neutrophil superoxide anion production following activation with FMLP (1027 M final concentration) as measured by isoluminol/HRP-amplified CL. The curves are from a representative experiment
and show the response induced in control cells (solid line) and exudated
cells (dashed line) isolated with a skin chamber technique. Abscissa, time
of study (minutes); ordinate, superoxide anion production (Mcpm).
Neutrophils exert their function in vivo mainly after extravasation, a process associated with a hierarchical mobilization of the
intracellular storage organelles. In exudated neutrophils, all secretory vesicles are mobilized together with ;40% of the gelatinase
granules and 20% of the specific granules (8). The extravasation
process thus results in an increased surface expression of various
receptors, including the FMLP-R (19), and as a consequence, such
cells were primed with respect to the NADPH-oxidase activity
induced by FMLP (Fig. 6).
In disagreement with the results obtained with ionomycintreated cells, the exudated neutrophils could be desensitized by
incubation with FMLP at 15°C (Fig. 7). The hypothesis stated
above was thus proved false.
Discussion
cesses of desensitization and termination of oxidase activity in
response to FMLP are not linked.
The mechanism for activation of the neutrophil NADPH-oxidase
via receptors for the N-formylated peptide FMLP has been shown
to involve a pertussis toxin-sensitive heterotrimeric G protein that
can pass the signal on to several transduction pathways working in
Termination of the FMLP-induced NADPH-oxidase activity
Many FMLP-induced responses (including activation of the
NADPH-oxidase) show transient kinetics. It has been suggested
that the rapid termination of the responses occurs through the desensitization brought on by the coupling of the receptor-ligand
complex to the cytoskeleton (14 –16). To investigate whether this
assumption holds true, we studied the kinetics (and thereby the
termination) of the response in the primed cell populations (ionomycin-treated and exudated cells, respectively) that differ in ability
to become desensitized.
We have shown earlier that the technique used to measure oxidase activity (isoluminol/HRP CL) is well suited for real time
studies of superoxide anion release from activated neutrophils
(21). In both ionomycin-treated and exudated cells, the FMLPinduced response was increased with respect to magnitude as well
as to the duration of the response (Figs. 1 and 6). However, the
kinetics of the production of superoxide anion was roughly the
same in these two cell populations, irrespective of whether the
cells were desensitizable (i.e., exudated cells) or not (i.e., ionomycin-treated cells) (Table I). Hence, our results show that the pro-
FIGURE 7. Desensitization of superoxide anion production in exudated neutrophils. Cells isolated with a skin chamber technique were incubated at 15°C in the presence (dashed line) or absence (solid line) of
FMLP (1027 M). The vials for measuring CL, containing HRP (4 U),
isoluminol (2 3 1025 M), and FMLP (1027 M), but no cells, were warmed
to 37°C in the luminometer. The cells were transferred from 15°C to the
measuring vials (37°C) at time 0, and the light production was recorded.
The curves are from a representative experiment. Abscissa, time of study
(minutes); ordinate, superoxide anion production (Mcpm).
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FIGURE 5. Lack of desensitization of the superoxide anion production
in ionomycin-treated neutrophils. The cells were pretreated with the calcium ionophore ionomycin (5 3 1027 M; 5 min), washed, and then incubated at 15°C in the presence (dashed line) or absence (solid line) of FMLP
(1027 M). The vials for measuring CL, containing HRP (4 U), isoluminol
(2 3 1025 M), and FMLP (1027 M), but no cells, were warmed to 37°C
in the luminometer. The cells were transferred from 15°C to the measuring
vials (37°C) at time 0, and the light production was recorded. The curves
are from a representative experiment. Abscissa, time of study (minutes);
ordinate, superoxide anion production (Mcpm).
The Journal of Immunology
2467
Table I. Characterization of the kinetics of the FMLP-induced CL
responses in the three neutrophil populations studied
Cell Populations
Control
a
Tmax
DTc
Integration/peakd
0.87 6 0.15
1.27 6 0.12
2.53 6 0.21
b
Exudated
Ionomycin-treated
1.05 6 0.07
2.10 6 0.14
2.80 6 0.14
1.10 6 0.10
1.93 6 0.15
3.37 6 0.29
a
Tmax represents the peak time (minutes) of the CL response.
Each numeral represents the mean 6 SD, n 5 3.
DT represents the time difference between the time point of the half-fall of the
CL response and the Tmax.
d
Integration/peak represents the integrated value of the CL response divided by
the peak CL value.
b
c
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parallel (10). In general, such signals are highly regulated, allowing cellular adaptation to a wide range of conditions. Desensitization is one such process (occurring in many cell types, including
the neutrophils), which is characterized by an absence of response
despite the presence of agonist-occupied receptors (26). A model
has been put forward stating that the desensitization process may
also be of importance for the termination of FMLP-induced neutrophil NADPH-oxidase activity (14 –16), and according to this
model, the production of second messengers ceases when the receptor-ligand complex binds to the cytoskeleton. We found that the
desensitization phenomenon was absent in ionomycin-primed
cells, and in accordance with the described model, the amount of
receptor-ligand complexes found to be associated with the cytoskeleton was decreased.
We know from earlier studies (22, 27, 28) that ionomycin induces a mobilization of neutrophil granules to the cell surface,
resulting in an increased exposure of different receptors. To measure the amount of FMLP receptors on ionomycin-primed cells,
FITC-labeled peptide was allowed to bind to the FMLP receptors,
and the cells were analyzed by FACS without prior washing. This
technique is superior to the technique previously used (both by us
and others), which was based on binding of radioactively labeled
peptides, since the latter required sedimentation and washing of the
cells to remove unbound peptide. This latter treatment caused a
partial detachment of bound ligand, resulting in an underestimation
of binding. Furthermore, the characteristics of the binding in that
system were such that it did not allow an extrapolation from the
binding data of receptor number and binding affinity (29 –31). The
receptor binding data that we obtained, showing a very low binding to ionomycin-treated cells, using a protocol with a radiolabeled
ligand and a brief washing procedure (not shown), and at the same
time a high level of binding with the FITC-labeled ligand, suggest
that the number of receptors are increased in these cells but that the
dissociation constant is higher compared with the nontreated cells.
Using high resolution binding data, it has been shown that the
FMLP-R may exist in two interconverting forms (32, 33), one low
affinity and one high affinity state. A detailed characterization of
the receptor conversion is beyond the specific aims of the present
work, which is focused on desensitization and termination of the
NADPH-oxidase; however, our results fit with the suggestion that
the conversion of the low affinity receptors to the high affinity state
is missing in ionomycin-treated cells.
The degree of receptor exposure was higher in ionomycinprimed cells (shown also for the CR3 molecules that are stored in
the same compartments as the FMLP-R (7)) compared with untreated control cells. We therefore conclude that the decrease in
number of ligand-receptor complexes recovered in the Triton
X-100-insoluble phase from ionomycin-primed cells is not due to
a decrease in receptor exposure but rather to an impaired association of the ligand-receptor complexes with the cytoskeleton.
One way to fit our data into the model of desensitization would
be to suggest that the newly mobilized receptors lack the ability to
become desensitized. To investigate this possibility, we determined the FMLP response in neutrophils that had exudated from
the blood stream, such cells having mobilized their intracellular
pools of receptors (8). The fact that the exudated cells could be
desensitized suggests that impaired desensitization is not due to a
recruitment of new, nondesensitizable receptors. In addition, our
results clearly show that the signals mediating desensitization are
also generated in exudated neutrophils, i.e., in cells that have experienced an intimate contact with the vascular endothelium and
the basement membrane and have been exposed to a large variety
of inflammatory mediators present in the exudate.
The molecular mechanisms behind the lack of desensitization in
ionomycin-primed cells can only be speculated upon at this time.
It has been shown that an occupation of the receptor for complement fragment 5a triggers an association of the FMLP-R to the
cytoskeleton (17), indicating that the association of these receptors
to the cytoskeleton is not a process driven by ligand-induced
changes of the receptor. It has been suggested that the desensitization is due to an actin-dependent/-driven segregation of the active ligand-receptor complex from the amplifying G protein(s). A
rise in intracellular Ca21 affects (directly or indirectly) the degree
of actin polymerization (Refs. 34 and 35; Fig. 3) as well as the
subcellular distribution of other cytoskeleton proteins (36, 37), and
an attractive explanation for the phenomenon observed in ionomycin-primed cells is that these changes secondarily interfere
with/affect the capability of the cytoskeleton to bind the occupied
receptors. At the present time, however, we can not exclude the
possibility that the receptors as well as the amplifying G protein(s)
are affected by the rise in intracellular calcium.
Binding of FMLP to its neutrophil surface receptors results in
superoxide anion production by the membrane-localized NADPHoxidase, but the active oxidase molecules are rapidly deactivated
(38). A sustained production of reactive oxygen species is thus a
result of continued production of second messengers, inducing a
replenishment of a small pool of active oxidase (38). According to
the hypothesis described above, association between the occupied
receptors and the cytoskeleton regulates the generation of activating signals and by that the termination of the oxidase activity (14 –
16, 25). We compared the time course of superoxide anion production in control cells and in the two cell populations that were
primed with respect to FMLP-induced generation of reactive oxygen species. The time required to reach the peak value was somewhat longer in the primed cells, as was the time for the half-fall of
the response (DT in Table I). However, the small changes in the
kinetics of the response were not related to the desensitization
phenomenon, since they occurred not only in ionomycin-treated
cells but also in exudated cells. Taken together, these data clearly
show that the termination of the oxidase response to FMLP is not
linked to the desensitization process.
Our results support the concept that an association of the ligandreceptor complex with the cytoskeleton is linked to a desensitization of the neutrophils with respect to activation of the oxidase.
Desensitization may be achieved through a direct interaction of the
receptor-ligand complex with cytoskeleton proteins such as actin
(16). A rise in intracellular Ca21 affects the organization of the
cytoskeleton (36, 37, 39, 40), and this should be expected to interfere with the desensitization process. With respect to the role of
desensitization in the termination of FMLP-induced oxidase activity, the results presented here strongly imply the existence of an
2468
LACK OF FMLP-R DESENSITIZATION IN IONOMYCIN-TREATED NEUTROPHILS
alternative, not yet identified mechanism that is responsible for the
termination of the oxidase activity.
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