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DOSE-DEPENDENCE
OF THE VARIOUS FUNCTIONAL RESPONSES
OF NEUTROPHILS TO FORMYLPEPTIDES
Activation,
Regulation, and Inverse Effects According to the Agonist Dose and CellCondition
P. BELLAVITE, S. CHIRUMBOLO, C. SANTONASTASO, D.
BIASI*, S. LUSSIGNOLI, G. ANDRIOLI
Institute of
Chimica Microscopia Clinica and of *Patologia Medica, University of Verona
Laboratorio
Centrale, Ospedale Policlinico, 37134 Verona, Italy
1 -Introduction
2 - Dose-response Curves of Activation, Priming and desensitization
3 - Other Dose-dependant Phenomena
4 - Inverse Effects of Different fMLP Doses on Neutrophil
Adhesion
5 - Discussion
6 - References
The up- and down-regulation of the response to a
specific stimulatory compound, according to its doses and to the sensitivity of
the cells, is a widespread process in biology, immunology and endocrinology.
Leukocytes are particularly suitable models for studying these changes of
biological response as these cells may be easily isolated from blood and from
inflammatory exudates and appear to be regulated by a large variety of
mediators both in vitro and in vivo (disease states).
Neutrophil
granulocytes are one of the main cell types involved in the first defense lines
against infections and in the acute inflammation. Following the local or
systemic generation of membrane perturbing agents and biological signals such
as cytokines, complement factors and bacterial products, neutrophils undergo a
series of highly regulated functional modifications. The expression and
activation of specific membrane glycoproteins induce cell adherence to
endothelium or to subendothelial structures, thus allowing the leukocyte
extravasation and the chemotactic movement into the connective tissue.
These
cells are endowed with a powerful armamentarium of enzymes and antimicrobial
peptides. Moreover, upon activation, they produce a huge amount of excited
oxygen species, including superoxide anion (O2-) and its derivatives, that
participate in the microbial killing but also in the possible tissue damage
caused by the dysfunction of neutrophil activation in a number of pathologies
(Weiss, 1989; Smith, 1994). Since the duration and the magnitude of the
functional responses are important factors in determining the final balance -
positive or unfavourable for the host - of neutrophil activation, these
phenomena are subject to a fine and complex regulation at the level of
receptors, transduction mechanisms and effector enzymes.
Here
we summarize a series of studies carried out in our laboratory with the aim of
characterizing the sensitivity limits of normal and primed human neutrophils to
the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP). We
studied: a) the oxidative metabolism, assessed as the burst of O2- production,
b) the adhesion to serum-coated surfaces, c) the release of lysozyme, a
bactericidal enzyme, d) the actin polymerization, a rapid agonist-stimulated
response of the cell cytoskeleton, e) the expression of fMLP receptors, f) the
activation of two important intracellular biochemical changes that follow
receptor stimulation, i.e. free Ca2+ increase and cyclic AMP (cAMP) increase.
These
data are of interest both for an understanding of neutrophil physiology in the
context of the inflammatory reactions and, in a wider perspective, for the
understanding of the complex events involved in the changes of sensitivity and
of responsiveness of the cells treated with different doses of receptor
agonists. Several experiments where the phenomena of direct activation,
priming, desensitization, and reverse effects have been observed are reported.
2. Dose-response
Curves of Activation, Priming and Desensitization
The dose-response curves of the effect of increasing
doses of fMLP on O2- production and on adhesion of human blood neutrophils are
shown in figure 1.
The
left panels of the figure (A and C) show the direct stimulatory effect of the agonist: O2- production was
triggered by fMLP doses of 10 nmoles (nM) and higher, reaching a maximum at 100
nM, while adhesion required at least 30 nM fMLP and increased up to 1000 nM.
The right panels of the figure (B and D) show the net effect of a second addition of high doses of fMLP (100 nM) to the cells
that had been previously treated with the range of fMLP concentrations
indicated in the X axis of the figure. It can be seen that the response of
neutrophils pre-treated with doses ranging from 0.1 to 5 nM (i.e.
sub-stimulatory doses) was higher than that of neutrophils not pre-treated with
fMLP, both in terms of O2- production (B) and in terms of adhesion (D).
Therefore, low doses of the peptide primed the cells to a subsequent higher
response to the same agent, a phenomenon that we called homologous priming (Bellavite et
al., 1993a). Figures 1B and 1D also show that the response of neutrophils pre-treated with doses ranging from
10 nM to 1000 nM (i.e. stimulatory doses as shown in the left panels of the
figure) had a decreased response both in terms of O2- production and in terms
of adhesion. In other words, the stimulatory doses of the peptide desensitize the cells to a subsequent
challenging with the same agent, and the extent of the desensitization is
proportional to the extent of the previous stimulation. Similar dose-dependent
stimulatory, priming and desensitising responses were observed by measuring the
release of lysozyme.
Priming
induced by low doses of fMLP is not only homologous but also heterologous,
because we and others have observed that pretreatment with fMLP increases also
the response to other unrelated agents, such as phorbol-myristate acetate
(PMA), the active principle of croton oil. On the other hand, the
desensitization induced by high doses of fMLP is exclusively homologous,
because the response to PMA was not inhibited but, instead, was further
augmented (Table 1).
3. Other
Dose-dependent Phenomena
We then investigated the possible mechanisms
underlying these low-dose (priming) and
high-dose (direct activation and
homologous desensitization) effects, by
looking at the correlation between these phenomena and other
fMLP-induced biochemical events.
Figure 1. O2- production (A, B) and adhesion (C, D) of human
neutrophils stimulated with different doses of fMLP. A, C: direct stimulation;
B, D: effect of 100 nM fMLP on cells pretreated for 15 minutes with the
indicated doses of fMLP. Blood neutrophils were obtained from healthy subjects
by centrifugation of EDTA-anticoagulated blood over Percoll gradients (Metcalf et al., 1986). O2- was measured by the reduction of ferricytochrome
c in multiwell microplates and adherence to the serum-coated surface of
microplates was quantitated by acid phosphatase assay (Bellavite et al., 1992). Values are mean ± S.D. of triplicate determinations from a
representative experiment of 12 performed.
TABLE 1. Effect of 15
minutes of pretreatment with different doses of fMLP on the O2- production induced by fMLP and PMA
__________________________________________________________
Preincubation O2- production (nmoles/106 cells/30 min)
__________________________________________
Second stimulus Second stimulus fMLP (100 nM) PMA
(10 ng/ml)
Buffer (control) 4.1 ± 0.5 13.6 ± 0.3
5 nM fMLP 10.1 ± 0.9 18.4 ± 1.3
100 nM fMLP 0.4 ± 0.4 35.5 ± 2.2
_________________________________________________________
Treatment
of neutrophils with 1 nM fMLP for 15 minutes raised the membrane fMLP binding
capacity by 60%, while treatment with
100 nM fMLP reduced the binding by over 90%, thus providing a clear molecular
explanation for the desensitization. Low
doses of fMLP (0.5 nM and up) were able to cause an
increase of intracellular free Ca2+ and of intracellular cAMP (Bellavite et al., 1993a,b; Biasi et al., 1993b). FMLP dose-response for
intracellular free Ca2+ was markedly
shifted to the left with respect to the dose response for O2- production: ED50 (dose causing 50% stimulation) were 1 nM for intracellular free
Ca2+ and approximately 50 nM for O2-
production respectively, indicating that the former is not a sufficient signal
for the activation of the oxidative metabolism.
Figure 2. Recording traces of right-angle light scatter response
(actin polymerization) of neutrophils to fMLP, assessed fluorimetrically
according to Omann et al. (1989).
These
data may indicate that the priming effect is the result of an increase in the
number of receptors or of other mechanisms of post-receptor regulation,
particularly linked to the increase of intracellular Ca2+. It should be noted,
however, that many models of priming exist and that an increase of Ca2+ does
not appear to be an absolute requirement (Biasi et al., 1993b). By no means extraneous to priming are other
biological events occurring at post-receptor level, such as the
phosphorylation-dephosphorylation of specific proteins, the state of assembly
of cytoskeletal proteins, and the constitution of membrane lipids. The latter
point appears to be particularly relevant for the in vivo regulation of neutrophil responsiveness, as documented by
our recent findings that the
O2- release in response to fMLP
is positively correlated with the cellular content of arachidonic acid and
inversely correlated with linoleic acid and palmitic acid content (Bellavite et al., 1995). These results suggest
that the fatty acid composition of blood neutrophils may be a critical factor
determining the capability of releasing free radicals in response to
formylpeptides and are of relevance in view of a possible manipulation of the
lipid composition of cell membranes by diet changes.
Another
cell response that appeared to be sensitive to very low doses of fMLP was the
actin polymerization, that is associated with cytoskeletal and morphological
changes of the cells (figure 2). Doses of fMLP as low as 0.2 nM triggered a
rapid actin polymerization response, that was already maximal at 20 nM. Of
particular interest are
also the time-dependent oscillations of scattering,
induced by medium-high doses of fMLP, a finding in agreement with previous ones
(Omann et al., 1989).
4. Inverse
Effects of Different fMLP Doses on Neutrophil Adhesion
We then studied the metabolic and adhesion responses
to fMLP of neutrophils which were treated with bacterial endotoxin
(lipopolysaccharide, LPS) or which were harvested from in a in vivo experimental inflammation (24-h
exudation through a skin-window procedure).
Both
LPS treatment and inflammation enhance the neutrophil's oxidative burst in
response to fMLP, i.e. they induce cell priming (table 2). Moreover, by investigating the dose-response
relationships of the adhesion response in these primed cells, we have observed
an unexpected phenomenon (table 2 and figure 3): a) priming augments cell
adhesion to serum-coated culture wells in the absence of further stimulation;
b) addition of low, substimulatory doses of fMLP (0.5 nM to 5 nM) inhibits and
reverses the spontaneous adhesion, c) high fMLP doses (> 100 nM) increase
the adhesion and are additive to the spontaneous adhesion induced by priming.
In conclusion, the chemotactic agent fMLP, which is considered to be an activator
of neutrophil adhesion, paradoxically inhibits the same cell response at low
doses when used in primed cells.
We have investigated several different neutrophil
functional activities triggered by contact with chemotactic peptides. The
evidence here reported demonstrates that "early" responses of
neutrophils to low doses of fMLP involve both structural (actin polymerization)
and biochemical (Ca2+ and cAMP increase)
changes, which are associated with inhibition of adhesion, while adhesion
and superoxide release should be
TABLE 2. A comparison of the
effects of low doses and high doses of fMLP on
O2- production and adhesion of normal and primed neutrophils
_________________________________________________________
Source of neutrophils O2- production (nmoles/106 cells/30 min)
_____________________________________
No fMLP Low-dose High-dose fMLPa fMLPb
Blood 0.6 ± 0.6 1.6
± 1.0 11.5 ± 3.0
LPS-primed 3.6 ± 0.3 2.8
± 0.3 18.9 ± 1.7
Exudate-primed 0.8 ± 1.0 5.4 ± 2.0 23.9
± 5.3
Adhesion (% of
total ) _____________________________________
No fMLP Low-dose
High-dose fMLPa fMLPb
Blood 5.6 ± 3.4 3.7
± 1.6 27.5 ± 8.1
LPS-primed 20.9 ± 10.5 7.4 ± 6.1 40.3 ± 16.9
Exudate-primed 17.1 ± 10.3 8.4 ±
4.9 39.2 ± 14.7
__________________________________________________________
Exudate neutrophils were
isolated according to the skin-window method described by Senn, with
modifications (Biasi et al., 1993a).
LPS-primed neutrophils were obtained by incubating blood neutrophils for 1h
with 1µg/ml LPS. a,b: Low doses 1 nM to 5 nM, high doses 100 nM to 500 nM,
according to different experiments. The table summarizes data from experiments
reported in Bellavite et al. (1993b)
and Bellavite et al. (1994). The
values significantly different (p < 0.01, paired Student t test) from the
respective values obtained in the absence of fMLP (no fMLP) are shown in bold.
Figure 3. Effect of different doses of fMLP on the adhesion of human
neutrophils. A: blood cells, B: cells isolated from a skin-window inflammatory
exudate of the same subject. Values are mean
± S.D. of triplicate determinations from a representative experiment of
12 performed.
considered as "late" or "extreme"
responses to chemotactic factors, which require a 100-fold higher dose of this
stimulant.
These
results indicate that actin polymerization, a typically non-linear cellular
phenomenon, reflects one of the earliest cellular events, occurring at very low
agonist doses. Our data are in agreement with those reported by others, showing
that half-optimal O2- production requires the occupancy of about 30% of the
fMLP receptors, while intracellular Ca2+ increase and actin polymerization
require occupancy of less than 3% and 0.1% receptors respectively (Sklar et al., 1985).
A summary of the various responses which have been
investigated and of their dependence on the fMLP dose is reported in table 3.
TABLE 3. Effects of
different doses of fMLP on various
neutrophil functions
_______________________________________________________________
Cell function Low doses High doses
(0.1 to 5 nM) (10 to 1000 nM)
_______________________________________________________________
Actin polymerization Activation Activation and
oscillations
Intracellular free Ca2+ Increase Increase
Intracellular cAMP Increase Increase
fMLP receptors Increase Decrease
Adhesion (normal blood
cells) No effect Activation
Adhesion (primed cells) Inhibition Activation
O2- production No effect Activation
Lysozyme release No effect Activation
_______________________________________________________________
These findings provide a reproducible in vitro model of the complex biological
events occurring in leukocytes when they are treated with different doses
of a bacterial product. In a wider
perspective, these findings provide an example of how biologically active
compounds may cause inverse effects on a system endowed with regulatory
(feed-back) controls, when either the doses
of the compound, or the sensitivity
and the responsiveness of the system
are varied by changes of environmental conditions. Neutrophil adhesion is the result of a fine regulation that
enables the cell to move onto a surface by continuous adhesion/detachment
events. Our current hypothesis is
that low doses of fMLP cause inhibition of cell adherence
because they increase cellular cAMP. In fact, adhesion is inhibited by
adenosine, a physiologically relevant agent that increases intracellular cAMP
(Bellavite et al., 1992). Moreover,
the adhesion of primed cells was inhibited also by addition of the membrane
permeating cAMP analogue dibutirryl cAMP (1 mM) and of theophyllin (2 mM),
which blocks the cAMP phosphodiesterase (Chirumbolo, unpublished observation).
The
increase of adhesion and the oxidative burst are induced by much high doses of
fMLP: it is conceivable that in order to trigger these responses, other
intracellular messengers, besides cAMP and calcium, are required. Candidate
molecules mediating the high-dose-dependent activation are diacylglycerols
generated by phospholipid breakdown, that are activators of protein kinase C
and whose production requires high fMLP doses (10 to 1000 nM) (Dougherty et al., 1989).
Our
data have shown the four different states of activation in neutrophils: a)
resting state (dormant cells); b) homologous and heterologous priming, with
increased O2- and decreased adhesion
responses; c) full activation, where the cells are treated with high doses of
agonists; d) specific desensitization to a second homologous stimulus. These
types of tests are not simple laboratory artefacts, but allow us to reproduce a
situation which occurs in vivo, i.e.
where the cells in patients presenting bacterial infections or systemic
inflammations are modified with respect to the cells in healthy people
(Bloomfield and Young, 1988; Trautinger et
al., 1991; Smith, 1994). The fact that the disease conditions cause changes
in receptor and transduction system sensitivity in various cells of the body is
well known in many fields of medicine.
The
concept is emerging that the leukocytes are involved in the cybernetic
information networks of inflammation in a highly sophisticated and complex way:
early activation of specific transduction systems, shape change, cell
detachment from the stores, priming and chemotaxis require the occupancy of a
minimum number of receptors, while the killing armamentarium (release of
granule constituents, oxygen radicals) and the potentially harmful cell
adhesion and spreading are triggered only when a massive engagement of membrane
receptors is achieved. Finally, the effects of endotoxin and of the cellular
lipid composition point to the existence of multiple control mechanisms and
subtle synergisms between endogenous and exogenous compounds. All these factors
are in connection with the individual's general state of health (infections,
neuro-immuno-endocrine equilibrium, nutritional status).
Acknowledgments.
This work was supported by grants from Guna s.r.l.
(Milano) and from Banca Popolare (Verona).
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